Molecular monolayers on surfaces are useful in many applications, including controlling corrosion, wetting or adhesion properties of a surface, performing heterogeneous catalysis, extracting or purifying analytes from solutions, and for producing biosensors or biochips.
A conventional process for depositing a molecules on a substrate surface involves immersing the surface in a solution having an excess of molecules for forming a self-completing monolayer on the surface. A disadvantage of conventional immersion based monolayer processing techniques is that they do not allow fine patterning. In addition, they are complex, expensive, slow, and inaccurate both chemically and geometrically. Conventional manufacture of DNA functionalized biochips involves spotting with DNA templates. Surface tension in the spot is relied upon to define spot geometry. Drying effects quickly change concentration in the spot and render control difficult.
Another conventional process for depositing molecules on a surface involves printing the monolayer on the surface from a stamp such as a stamp made in poly dimethyl siloxane (PDMS). Printing allows patterning of the surface with minute amounts of molecules. However, there is a variable transfer associated with such printing. Coverage at a given spot on the surface depends on both inking density and printing efficiency. This is particularly problematical where molecules can only be placed as a monolayer on the stamp in, for example, printing of catalysts or biological molecules. Here, contrast between a passive and a functional surface is provided by the presence or absence of a single molecule. A single missing molecule produces a defect. Conventionally, transfer of monolayers from a stamp to a surface is not free of defects and transfer ratios vary from one print cycle to the next. This is undesirable for mass production environments. See, for example, A. Bernard et al., “Microcontact Printing of Proteins”, Adv. Mater. 2000 (12), 1067 (2000)
So-called biochip or micro array technology is increasingly important in applications such as genetic analysis, including examination of gene activity and identification of gene mutations. Genetic information can be used to improve drug screening, diagnostics, medication, and identification. A typical biochip for such an application comprises a miniature array of gene fragments or proteins attached to a glass surface. Typically a hybridization reaction between sequences on the surface of such biochips and a fluorescent sample is used for the analysis. Following hybridization, biochips are typically read with fluorescence detectors, permitting the fluorescent intensity of spots on the surface to be quantified. Protein markers, viruses, and protein expression profiles can be similarly detected via protein specific capture agents. Conventional methods for patterning biological molecules on biochips are described in M. Schena, “Micro array Biochip Technology”, Eaton Publishing, Natick Mass., (2000) and G. Ramsay, “DNA chips: State of the Art”, Nature Biotech, 16, 40 (1998). Conventional methods include sequential and parallel patterning techniques. The sequential techniques serially address spots on the surface. These techniques include: pipetting; capillary printing; ink jet printing; and, pin spotting. The parallel techniques pattern multiple areas of molecules onto the surface simultaneously. These techniques include: microfluidic network delivery; capillary array printing; and, microcontact printing. Microcontact printing involves inking a patterned stamp. Such inking may be performed via a microfluidic network.
Deoxyribonucleic acid (DNA) may be applied to a biochip surface for some applications. The information encoded in DNA establishes and maintains cellular and biochemical functions of an organism. In most organisms, DNA is an extended double stranded polymer. The sequence of deoxyribonucleotides of one DNA is complementary to those of the other strand. This enables new DNA molecules to be synthesized with the same linear array of deoxyribonucleotides in each strand as an original DNA molecule. This process is generally referred to as DNA replication. The DNA code is made up from four bases: adenine (A), guanine (G), cytosine (C), and thymine (T). A nucleotide consists of one of the four organic bases, a five carbon sugar (pentose), and a phosphate group. The phosphate group and organic base are attached to the 5′ carbon and 1′ carbon atoms of the sugar moiety, respectively. The sugar of DNA is 2′ deoxyribose because it has a hydroxyl group only on the 3′ carbon. The nucleotides of DNA are joined by phoshodiester bonds with the phosphate group of the 5′ carbon of one nucleotide linked to the 3′ OH of the deoxyribose of the sugar of the adjacent nucleotide. A polynucleotide thus has a 3′ OH at one end (3′end) and a 5′ phosphate group at the other end (5′end). DNA forms a double stranded helix with bases A pairing with T and bases G pairing with C via two and three hydrogen bonds, respectively. The two strands of a duplex DNA run in opposite directions, by convention double stranded DNAs are always written with the 5′ end of the upper strand on the left. During the enzymatic replication process, the phosphate of the added nucleotide is linked to the 3′ OH of the existing sequence. Thus, DNA is always replicated from 5′ to 3′ direction as described in B. R. Glick, et al., “Molecular Biotechnology: Principles and Applications of Recombinant DNA”, American Society for Microbiology, Washington 1998.
Manufacture of DNA functionalized biochips conventionally involves sequential inking of spots with a different DNA template to from DNA targets. This is complex, slow and thus expensive process.
Conventionally, gene analysis was performed by hybridization of labeled probes to the DNA targets that were passively adsorbed to support surfaces such as nitrocellulose, nylon membranes, or lysine coated glass slides. Covalent linkage of DNA to the surface provides stable attachment under hybridization conditions. DNA oligomers can be attached or synthesized in situ from either the 3′-end or the 5′-end. Processes for attaching 5′-end oligonucleotides to glass include: an epoxy opening reaction on epoxy silane derivatized glass such as described in K. L. Beattie et al. Clin. Chem. 41, 700-706 (1995); 5′-succinylated target oligonucleotides immobilized onto amino derivatized glass such as described in Joos, B. et al., Anal. Biochem. 247, 96-101 (1997); and, 5′-disulfide modified oligonucleotides bound via disulfide bonds onto thiol derivatized glass such as described in Rogers, Y. H. et al., Anal. Biochem. 266, 23-30 (1999). Other processes use cross linkers such as pehyldiisocyanate, maleic anhydride, or carbodiimides and are described, for example, in Chrisey, L. A. et al., Nucleic Acids Res. 24, 3031-3039 (1996), O'Donnell, M. J. et al., Anal. Chem. 69, 2438-2443 (1997), Chee, M., et al., “Accessing genetic information with high-density DNA arrays”, Science 274, 610-614 (1996). An overview of attachment chemistries is published in G. T. Hermanson, “Bioconiugate Techniques”, Academic Press, San Diego, 1996. Reproducible chemisorption of oligomers in particular are described in: Adessi, C. et al. Nucleic Acid Res. 28 (e87) 1-8 (2000); Kawashima, E., et al., “Method of nucleic acid amplification”, WO 98/44151; and, Adessi, C., et al., “Methods of nucleic acid amplification and sequencing”, WO 00/18957.
The polymerase chain reaction (PCR) is an in vitro technique permitting exponential amplification of a specific ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) region lying between two regions of known DNA sequence. Conventional applications of PCR include: gene characterization; cloning; DNA diagnostics for pathogen detection; identifying mutations responsible for inherited diseases; and, DNA fingerprinting. PCR amplification is achieved via oligonucleotide primers known as ampliprimers. These are short, single stranded DNA molecules which are complementary to the ends of a defined sequence of DNA template. The primers are extended in 3′ directions on single stranded denatured DNA by a thermostable DNA polymerase in the presence of deoxynucleoside triphosphates (dNTPs) under suitable reaction conditions. Strand synthesis can be repeated by heat denaturation of the double stranded DNA, annealing of primers by cooling the mixture and primer extension by DNA polymerase at a temperature suitable for enzyme reaction. Each repetition of strand synthesis comprises a cycle of amplification. Each new DNA strand synthesized becomes a template for any further cycle of amplification. The amplified target DNA is thus amplified exponentially. For further information relating to PCR, see C. R. Newton et al., “PCR”, Bios Scientific Publishers, Oxon, U. K. 2000, E. Southern et al. “Molecular Interactions on Microarrays”, Nature Genetics 21, 5 (1999); U. Maskos et al., “Oligonucleotide hybridizations on glass supports” Nucleic Acid Res. 20(7), 1679-1684 (1992); Z. Guo et al., “Direct Fluorescence Analysis of Genetic Polymorphisms by Hybridization with Oligonucleotide Arrays on Glass Supports” Nucleic Acid Res. 22 (24), 5456-5465 (1994); J. Lamture et al., “Direct Fluorescence of Nucleic Acid Hybridization on the Surface of a Charge Coupled Device” Nucleic Acid Res. 22(11), 2121-2125 (1994); M. Sjoeroos et al., “Solid-Phase PCR with Hybridization and Time-Resolved Fluorometry for Detection of HLA-B27”, Clinical Chem. 47(3), 498 (2001).